The present invention is directed toward a novel process for producing a purified cross-linked hemoglobin product suitable as a blood replacement for transfusion in human beings and in animals or as an oxygen transport fluid. The subject cross-linked hemoglobin product is essentially free of contaminants in that it contains very low levels of bacterial endotoxin and phospholipid.
The structure and function of hemoglobin have been reviewed (Bunn, H. F., and B. G. Forget, Hemoglobin: Molecular, Genetic and Clinical Aspects, W. B. Saunders Company, Philadelphia, 690 pp., (1986). Mammalian hemoglobin is a tetramer containing two each of two different types of subunits, alpha and beta. Each subunit is of approximate molecular weight 16,000 and contains a heme group with a central iron atom. The protein portion, or globin functions to keep the heme iron in the reduced or ferrous state, thus to allow the hemoglobin molecule to reversibly bind oxygen.
Mammalian hemoglobin should not be thought of as a static tetramer, that is a single protein species with a molecular weight of 64-68,000. It is instead composed of sub-units connected by noncovalent bonds, and existing in dynamic equilibria involving monomer-dimer and dimer-tetramer interactions. At any moment, the hemoglobin would consist of a certain proportion of monomers with a molecular weight of approximately 16,000, dimers of molecular weight 32,000, and tetramers. The distribution of monomer, dimer, and tetramer species in solution are dependent upon hemoglobin concentration, the ligand state of the hemoglobin, as well as pH and salt composition of the solution in which it is contained.
Mammalian hemoglobin is contained within red blood cells or erythrocytes. In the mammals these specialized cells have lost their nuclei during maturation, and are simply sacs of hemoglobin containing very low concentrations of other proteins. The hemoglobin is contained with circulating erythrocytes for several reasons. First of all, if the hemoglobin were not in cells but instead circulated free in solution, it would readily be cleared from circulation by passage through the capillary walls, the kidney and other sites. Certain invertebrate organisms have solved this problem by evolving polymeric hemoglobins with molecular weights in the millions which are too large to pass through capillaries and the nephron of the kidney. Other functions served by the red cell are to provide a sheltered environment to protect the hemoglobin molecule from proteolytic serum proteins, maintain a balance of ions proper for hemoglobin function, provide an enzyme system which maintains the heme iron in the reduced (functional) state, and control the presence of allosteric effector molecules.
There have been numerous efforts to produce clarified stroma-free hemoglobin solutions for blood replacement transfusion in humans (Pennell, R. B., and W. E. Smith, Preparation of stabilized solutions of hemoglobin. Blood 4:380-385, (1949); Rabiner, S. F., Helbert, J. R., Lopas, H., and L. H. Friedman, Evaluation of a strom-free hemoglobin solution for use as a plasma expander. J. Exp. Med. 126: 1127-1142 (1967); Christensen, S. M., Medina, F., Winslow, R. W., Snell, S. M., Zegna, A., and M. A. Marini, Preparation of human hemoglobin Ao for possible use as a blood substitute. J. Biochem. Biophys. Meth. 17: 143-154 (1988); and Feola, M., Gonzalez, H., Canizaro, P. C., Bingham, D. and P. Periman, Development of a bovine stroma-free hemoglobin solution as a blood substitute. Surg. Gyn. Obst. 157: 399-408 (1983)). These solutions contain the hemoglobin in an unmodified state, and the hemoglobin is free to dissociate into its subunits. Infusion of unmodified hemoglobin leads to rapid clearance of the hemoglobin from the circulation. In addition, infusion of unmodified hemoglobin reportedly leads to a host of deleterious effects within the body including nephrotoxicity. The nephrotoxicity associated with unmodified hemoglobin has prompted the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration to state that unmodified hemoglobin should not be present in a hemoglobin-based oxygen carrier.
There have been numerous reports describing the stabilization of hemoglobin solutions by forming covalent chemical cross-links between the hemoglobin polypeptide chains (Rausch, C. W., and M. Feola, Extra pure semi-synthetic blood substitute, International patent application No. PCT/US87/02967, Int. publication no. WO/88/03408 19 May 1988; Bonhard, K., and N. Kothe Cross-linked hemoglobin of extended shelf life and high oxygen transport capacity and process for preparing same, U.S. Pat. No. 4,777,244; Bucci. E., Razynska, A., Urbaitis, B., and C. Fronticelli Pseudo cross-link of human hemoglobin with mono-(3,5-dibromosalicyl)fumarate. J. Biol. Chem. 264:6191-6195 (1989); and U.S. Pat. No. 4,001,200.)